Upload
anon908384753
View
118
Download
3
Tags:
Embed Size (px)
Citation preview
NITRILASE ASSAYS
Ashlesha Bhondwe
INTRODUCTION
Nitrilases are enzymes that catalyse the hydrolysis of organontiriles to the corresponding carboxylic acids and ammonia.
NITRILASES
Play an important role in disposing off toxic nitriles.
They are regioselective enzymes. Play an important role in the synthesis of pharmaceutical intermediates.
These enzymes are classified based on their substrate specificity as aromatic or aliphatic nitrilases.
MECHANISM OF THE ENZYME
METHODS FOR SCREENING OF NITRILASE ACTIVITY
High throughput microbial nitrilase screening are necessary .
Techniques such as HPLC, mass spectroscopy are time consuming.
A high throughput amenable colorimetric assay for enantioselective screening of nitrilase producing micro organisms using pH sensitive Indicators.(Banerjee et.al, 2003)
pH drop determined due to formation of acids
Material used for the assay: •Bromothymol blue 0.01%•Phosphate Buffer (10mM, pH- 7.2)•Mandelonitrile (500mM stock)
R- CN R-COO- + H+
pKa of indicator – 7.2 (similar to the pKa of the buffer)
Proportionality between the rate of change of absorbance of indicator and rate of the reaction, given by buffer factor, Q
Yellow
Bromothym
ol
Blue
CALCULATION
Q= (Cb / Cin )* (1/Δ ε*l)
Q= Buffer FactorCb = concentration of buffer
Cin = concentration of indicator
Δ ε = molar extinction coefficient
Rate (µmol/min) = (dA/dt) * Q
ASSAY
Assay carried out in a 96 well microplate for rapid screening of microorganisms. Absorbance at 616nm
The decrease in absorbance is proprtional to the concentration of proton released.
A Metal ion based method for screening of Nitrilases.
(Daniel R et.al, 2006)
Metal ion cobalt was used to determine the amount of ammonia released by the action of nitrilase.
ASSAY
Material used for assay : BufferedCobalt chloride (10mM) – made in
10mM Tris pH- 7. Substrate – hydrocinnamonitrile and
isobutylsuccinonitrile Reaction quenched by increasing the
temperature from 30 ° C to 95° C Assay validated by HPLC. As soon as the cobalt chloride solution is
added to the reaction mixture a yellow colored complex is formed. Absorbance at 375nm.
LIMITATION
This technique has a high background interference due to the initial colour of cobalt chloride.
For this reason higher concentrations of ammonia can be detected (above 5mM).